Minutes are small, spherical, extra-chromosonal bodies, observed in pairs (double minutes or DMs) during metaphase in certain cell types (transformed, viral infected or mycoplasma infected cells or cells exposed to certain types of radiation). The evidence indicates they are related to another intra-chromosomal aberration (homogeneously staining regions or HSRs), carry active genes, are the result of a gene amplification mechanism and autonomously replicate their DNA. The amount of DNA contained in DMs has been approximated in various ways (but never physically measured) at around 6 x 10 to the 8 daltons, a mass for larger than that required to amplify most genes (mass about 3 x 10 to the 7). The proposal develops the working hypothesis that a unit of gene amplification, the replicon cluster and the single minute are all different manifestations of a chromosomal subunit of structure. The research will investigate DNA replication in the minute and certain aspects of its structure (from at least one cell source), including a physical determination of mass (by sedimentation and electron microscopy), determination of base sequence homogeneity between minutes within a cell population (restriction nuclease digestion of DNA followed by gel electrophoresis of the fragments) and an examination of the intact and histone-depleted structure to detect associations between structure and DNA replication (scanning and transmission electron microscopy and EM autoradiography). Other experiments will study the proteins associated with intact and histone depleted structures (gel electrophoresis) and look for structural similarities between DMs and HSR regions (EM). Longer term objectives presently include looking for analogies between minute structure and that of the normal mammalian chromosome, and using the minute as a source of (potentially) uniform replication-origin base sequences.